DOI QR코드

DOI QR Code

The application of new breeding technology based on gene editing in pig industry - A review

  • Tu, Ching-Fu (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute) ;
  • Chuang, Chin-kai (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute) ;
  • Yang, Tien-Shuh (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute)
  • Received : 2021.09.03
  • Accepted : 2021.12.07
  • Published : 2022.06.01

Abstract

Genome/gene-editing (GE) techniques, characterized by a low technological barrier, high efficiency, and broad application among organisms, are now being employed not only in medical science but also in agriculture/veterinary science. Different engineered CRISPR/Cas9s have been identified to expand the application of this technology. In pig production, GE is a precise new breeding technology (NBT), and promising outcomes in improving economic traits, such as growth, lean or healthy meat production, animal welfare, and disease resistance, have already been documented and reviewed. These promising achievements in porcine gene editing, including the Myostatin gene knockout (KO) in indigenous breeds to improve lean meat production, the uncoupling protein 1 (UCP1) gene knock-in to enhance piglet thermogenesis and survival under cold stress, the generation of GGTA1 and CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene double KO (dKO) pigs to produce healthy red meat, and the KO or deletion of exon 7 of the CD163 gene to confer resistance to porcine reproductive and respiratory syndrome virus infection, are described in the present article. Other related approaches for such purposes are also discussed. The current trend of global regulations or legislation for GE organisms is that they are exempted from classification as genetically modified organisms (GMOs) if no exogenes are integrated into the genome, according to product-based and not process-based methods. Moreover, an updated case study in the EU showed that current GMO legislation is not fit for purpose in term of NBTs, which contribute to the objectives of the EU's Green Deal and biodiversity strategies and even meet the United Nations' sustainable development goals for a more resilient and sustainable agri-food system. The GE pigs generated via NBT will be exempted from classification as GMOs, and their global valorization and commercialization can be foreseen.

Keywords

Acknowledgement

This article was financially supported by the Council of Agriculture, Executive Yuan, Taiwan ROC, through project 110AS-1.4.3-AD-U1.

References

  1. Craft WA. Fifty years of progress in swine breeding. J Anim Sci 1958;17:960-80. https://doi.org/10.2527/jas1958.174960x
  2. Knap PW, Rauw WM. Selection for high production in pigs. In: Rauw W, editor. Resource allocation theory applied to farm animal production. Wallingford, UK: CABI International; 2009. pp. 210-29.
  3. Yang TS. Wild to domestic: body and organ size matter fitness in boars. In: Jenkins OP, editor. Advances in zoology research. NY, USA: Nova Science Publishers, Inc.; 2012. p. 187-200.
  4. Merks JWM, Mathur PK, Knol EF. New phenotypes for new breeding goals in pigs. Animal 2012;6:4,535-43. https://doi.org/10.1017/S1751731111002266
  5. Hermesch S, Li L, Doeschl-Wilson AB, Gilbert H. Selection for productivity and robustness traits in pigs. Anim Prod Sci 2015;55:1437-47. https://doi.org/10.1071/AN15275
  6. Ryu J, Prather RS, Lee K. Use of gene-editing technology to introduce targeted modifications in pigs. J Anim Sci Biotechnol 2018;9:5. https://doi.org/10.1186/s40104-017-0228-7
  7. Yang H, Wu Z. Genome editing of pigs for agriculture and biomedicine. Front Genet 2018;9:360. https://doi.org/10.3389/fgene.2018.00360
  8. Zhao J, Lai L, Ji W, Zhou Q. Genome editing in large animals: current status and future prospects. Natl Sci Rev 2019;6:402-20. https://doi.org/10.1093/nsr/nwz013
  9. Penrith ML, Bastos A, Chenais E. With or without a vaccine-A review of complementary and alternative approaches to managing african swine fever in resource-constrained smallholder settings. Vaccines 2021;9:116. https://doi.org/10.3390/vaccines9020116
  10. Hansen PJ. Prospects for gene introgression or gene editing as a strategy for reduction of the impact of heat stress on production and reproduction in cattle. Theriogenology 2020;154:190-202. https://doi.org/10.1016/j.theriogenology.2020.05.010
  11. Tanihara F, Hirata M, Otoi T. Current status of the application of gene editing in pigs. J Reprod Dev 2021;67:177-87. https://doi.org/10.1262/jrd.2021-025
  12. Zhang J, Khazalwa EM, Abkallo HM, et al. The advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research. J Genet Genomics 2021;48:347-60. https://doi.org/10.1016/j.jgg.2021.03.015
  13. Petersen GEL, Buntjer J, Hely FS, Byrne TJ, Whitelaw B, Doeschl-Wilson A. Gene editing in Farm Animals: A Step Change for Eliminating Epidemics on our Doorstep? bioRxiv 2021 April 19 [preprint]. https://doi.org/10.1101/2021.04.19.440533
  14. Moehle EA, Rock JM, Lee YL, et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci USA 2007;104:3055-60. https://doi.org/10.1073/pnas.0611478104
  15. Christian M, Cermak T, Doyle EL, et al. Targeting DNA doublestrand breaks with TAL effector nucleases. Genetics 2010;186:757-61. https://doi.org/10.1534/genetics.110.120717
  16. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339:819-23. https://doi.org/10.1126/science.1231143
  17. Stoddard BL. Homing endonucleases: from microbial genetic invaders to reagents for targeted DNA modification. Structure 2011;19:7-15. https://doi.org/10.1016/j.str.2010.12.003
  18. Smith J, Bibikova M, Whitby FG, Reddy AR, Chandrasegaran S, Carroll D. Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res 2000;28:3361-9. https://doi.org/10.1093/nar/28.17.3361
  19. Bibikova M, Carroll D, Segal DJ, et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 2001;21:289-97. https://doi.org/10.1128/MCB.21.1.289-297.2001
  20. Streubel J, Blucher C, Landgraf A, Boch J. TAL effector RVD specificities and efficiencies. Nat Biotechnol 2012;30:593-5. https://doi.org/10.1038/nbt.2304
  21. Richter A, Streubel J, Boch J. TAL effector DNA-binding principles and specificity. In: Kuhn R, Wurst W, Wefers B, editors. Methods in Molecular Biology, vol 1338. New York, NY, USA: Humana Press; 2016. pp. 9-25. https://doi.org/10.1007/978-1-4939-2932-0_2
  22. Cermak T, Doyle EL, Christian M, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA14 targeting. Nucleic Acids Res 2011;39:e82. https://doi.org/10.1093/nar/gkr218
  23. Cermak T, Starker CG, Voytas DF. Efficient design and assembly of custom TALENs using the golden gate platform. In: Pruett-Miller S, editor. Chromosomal mutagenesis. Methods in molecular biology (methods and protocols), vol 1239. New York, NY, USA: Humana Press; 2015. pp. 133-59. https://doi.org/10.1007/978-1-4939-1862-1_7
  24. Morbitzer R, Elsaesser J, Hausner J, Lahaye T. Assembly of custom TALE-type DNA binding domains by modular cloning. Nucleic Acids Res 2011;39:5790-9. https://doi.org/10.1093/nar/gkr151
  25. Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol 2011;29:149-53. https://doi.org/10.1038/nbt.1775
  26. Deng P, Carter S, Fink K. Design, construction, and application of transcription activation-like effectors. In: Manfredsson F, Benskey M, editors. Viral vectors for gene therapy. Methods in molecular biology, vol 1937. New York, NY, USA: Humana Press; 2019. pp. 47-58. https://doi.org/10.1007/978-1-4939-9065-8_3
  27. Nitsch S, Mussolino C. Generation of TALE-based designer epigenome modifiers. In: Jeltsch A, Rots M, editors. Epigenome editing. Methods in molecular biology, vol 1767. New York, NY, USA: Humana Press; 2018. pp. 89-109. https://doi.org/10.1007/978-1-4939-7774-1_4
  28. Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337:816-21. https://doi.org/10.1126/science.1225829
  29. Josephs EA, Kocak DD, Fitzgibbon CJ, et al. Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage. Nucleic Acids Res 2015;43:8924-41. https://doi.org/10.1093/nar/gkv892
  30. Sternberg SH, LaFrance B, Kaplan M, Doudna JA. Conformational control of DNA target cleavage by CRISPR-Cas9. Nature 2015;527:110-3. https://doi.org/10.1038/nature15544
  31. Lee K, Uh K, Farrell K. Current progress of genome editing in livestock. Theriogenology 2020;150:229-35. https://doi.org/10.1016/j.theriogenology.2020.01.036
  32. Nishimasu H, Ran FA, Hsu PD, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 2014;156:935-49. https://doi.org/10.1016/j.cell.2014.02.001
  33. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without doublestranded DNA cleavage. Nature 2016;533:420-4. https://doi.org/10.1038/nature17946
  34. Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage. Nature 2017;551:464-71. https://doi.org/10.1038/nature24644
  35. Richter MF, Zhao KT, Eton E, et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol 2020;38:883-91. https://doi.org/10.1038/s41587-020-0453-z
  36. Komor AC, Zhao KT, Packer MS, et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv 2017;3:eaao4774. https://doi.org/10.1126/sciadv.aao4774
  37. Anzalone AV, Randolph PB, Davis JR, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019;576:149-57. https://doi.org/10.1038/s41586-019-1711-4
  38. Marzec M, Braszewska-Zalewska A, Hensel G. Prime editing: a new way for genome editing. Trends Cell Biol 2020;30:257-9. https://doi.org/10.1016/j.tcb.2020.01.004
  39. Grobet L, Martin LJ, Poncelet D, et al. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat Genet 1997;17:71-4. https://doi.org/10.1038/ng0997-71
  40. Grobet L, Poncelet D, Royo LJ, et al. Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle. Mamm Genome 1998;9:210-3. https://doi.org/10.1007/s003359900727
  41. Czaja W, Nakamura YK, Li N, et al. Myostatin regulates pituitary development and hepatic IGF1. Am J Physiol Endocrinol Metab 2019;316:E1036-49. https://doi.org/10.1152/ajpendo.00001.2019
  42. Qian L, Tang M, Yang J, et al. Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs. Sci Rep 2015;5:14435. https://doi.org/10.1038/srep14435
  43. Wang K, Ouyang H, Xie Z, et al. Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system. Sci Rep 2015;5:16623. https://doi.org/10.1038/srep16623
  44. Wang K, Tang X, Liu Y, et al. Efficient generation of orthologous point mutations in pigs via CRISPR-assisted ssODN-mediated homology-directed repair. Mol Ther Nucleic Acids 2016;5:e396. https://doi.org/10.1038/mtna.2016.101
  45. Wang K, Tang X, Xie Z, et al. CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs. Transgenic Res 2017;26:799-805. https://doi.org/10.1007/s11248-017-0044-z
  46. Zou YL, Li ZY, Zou YJ, et al. Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination. J Integr Agric 2019;18:1329-36. https://doi.org/10.1016/S2095-3119(19)62694-8
  47. Paek HJ, Luo ZB, Choe HM, et al. Association of myostatin deficiency with collagen related disease-umbilical hernia and tippy toe standing in pigs. Transgenic Res 2021;30:663-74. https://doi.org/10.1007/s11248-021-00275-6
  48. Xiang G, Ren J, Hai T, et al. Editing porcine IGF2 regulatory element improved meat production in Chinese Bama pigs. Cell Mol Life Sci 2018;75:4619-28. https://doi.org/10.1007/s00018-018-2917-6
  49. Li R, Zeng W, Ma M, et al. Precise editing of myostatin signal peptide by CRISPR/Cas9 increases the muscle mass of Liang Guang Small Spotted pigs. Transgenic Res 2020;29:149-63. https://doi.org/10.1007/s11248-020-00188-w
  50. Shi I, Luo L, Eash J, Ibebunjo C, Glass DJ. The SCF-Fbxo40 complex induces IRS1 ubiquitination in skeletal muscle, limiting IGF1 signaling. Dev Cell 2011;21:835-47. https://doi.org/10.1016/j.devcel.2011.09.011
  51. Zou Y, Li Z, Zou Y, Hao H, Li N, Li Q. An FBXO40 knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects. Biochem Biophys Res Commun 2018;498:940-5. https://doi.org/10.1016/j.bbrc.2018.03.085
  52. Zhu X, Wei Y, Zhan Q, et al. CRISPR/Cas9- mediated biallelic knockout of IRX3 reduces the production and survival of somatic cell-cloned Bama minipigs. Animals 2020;10:501. https://doi.org/10.3390/ani10030501
  53. Hou L, Shi J, Cao L, Xu G, Hu C, Wang C. Pig has no uncoupling protein 1. Biochem Biophys Res Commun 2017;487:795-800. https://doi.org/10.1016/j.bbrc.2017.04.118
  54. Trayhurn P, Temple NJ, Van Aerde J. Evidence from immunoblotting studies on uncoupling protein that brown adipose tissue is not present in the domestic pig. Can J Physiol Pharmacol 1989;67:1480-5. https://doi.org/10.1139/y89-239
  55. Berg F, Gustafson U, Andersson L. The uncoupling protein 1 gene (UCP1) is disrupted in the pig lineage: A genetic explanation for poor thermoregulation in piglets. PLoS Genet 2006;2:e129. https://doi.org/10.1371/journal.pgen.0020129
  56. Lin J, Cao C, Tao C, et al. Cold adaptation in pigs depends on UCP3 in beige adipocytes. J Mol Cell Biol 2017;9:364-75. https://doi.org/10.1093/jmcb/mjx018
  57. Zhang Q, Lin J, Huang J, et al. Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity. Proc Natl Acad Sci USA 2017;114:E9474-82. https://doi.org/10.1073/pnas.1707853114
  58. Commins SP, Jerath MR, Cox K, Erickson LD, Platts-Mills T. Delayed anaphylaxis to alpha-gal, an oligosaccharide in mammalian meat. Allergol Int 2016;65:16-20. https://doi.org/10.1016/j.alit.2015.10.001
  59. de la Fuente J, Cabezas-Cruz A, Pacheco I. Alpha-gal syndrome: challenges to understanding sensitization and clinical reactions to alpha-gal. Expert Rev Mol Diagn 2020;20:905-11. https://doi.org/10.1080/14737159.2020.1792781
  60. U. S. Food and Drug Admin. FDA Approves first-of-its-kind intentional genomic alteration in line of domestic pigs for both human food, potential therapeutic uses [internet]. c2020 [2020 Dec 14]. Available from: https://www.fda.gov/newsevents/press-announcements/fda-approves-first-its-kindintentional-genomic-alteration-line-domestic-pigs-bothhuman-food
  61. Jahan M, Thomsona PC, Wynna PC, Wang B. The nonhuman glycan, N-glycolylneuraminic acid (Neu5Gc), is not expressed in all organs and skeletal muscles of nine animal species. Food Chem 2021;343:128439. https://doi.org/10.1016/j.foodchem.2020.128439
  62. Tangvoranuntakul P, Gagneux P, Diaz S, et al. Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. Proc Natl Acad Sci USA 2003;100:12045-50. https://doi.org/10.1073/pnas.2131556100
  63. Alisson-Silva F, Kawanishi K, Varki A. Human risk of diseases associated with red meat intake: Analysis of current theories and proposed role for metabolic incorporation of a nonhuman sialic acid. Mol Aspects Med 2016;51:16-30. https://doi.org/10.1016/j.mam.2016.07.002
  64. Chuang Ck, Chen CH, Huang CL, et al. Generation of GGTA1 mutant pigs by direct pronuclear microinjection of CRISPR/Cas9 plasmid vectors. Anim Biotechnol 2017;28:174-81. https://doi.org/10.1080/10495398.2016.1246453
  65. Tu CF, Chuang CK, Hsiao KH, et al. Lessening of porcine epidemic diarrhoea virus susceptibility in piglets after editing of the CMP-N-glycolylneuraminic acid hydroxylase gene with CRISPR/Cas9 to nullify N-glycolylneuraminic acid expression. PLoS One 2019;14:e0217236. https://doi.org/10.1371/journal.pone.0217236
  66. Yen CH, Tai HC, Peng SH, Yang TS, Tu CF. Scaffold derived from GGTA1 and CMAH double knockout pigs elicits only slight inflammation in a gene-edited pig model. Materialia 2020;14:100836. https://doi.org/10.1016/j.mtla.2020.100836
  67. Ghosh D, Kumar A, Sinha N. Targeted genome editing: a new era in molecular biology. In: Editors: Mondal S and Singh RL, editors. Advances in animal genomics. London, UK: Elsevier Inc.; 2021. pp. 75-89. https://doi.org/10.1016/B978-0-12-820595-2.00006-0
  68. You W, Li M, Qi Y, et al. CRISPR/Cas9-mediated specific integration of Fat-1 and IGF-1 at the pRosa26 locus. Genes 2021;12:1027. https://doi.org/10.3390/genes12071027
  69. Gu H, Zhou Y, Yang J, et al. Targeted overexpression of PPARγ in skeletal muscle by random insertion and CRISPR/Cas9 transgenic pig cloning enhances oxidative fiber formation and intramuscular fat deposition. FASEB J 2021;35:e21308. https://doi.org/10.1096/fj.202001812RR
  70. Whitworth KM, Lee K, Benne JA, et al. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol Reprod 2014;91:78. https://doi.org/10.1095/biolreprod.114.121723
  71. Burkard C, Lillico SG, Reid E, et al. Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLoS Pathogens 2017;13:e1006206. https://doi.org/10.1371/journal.ppat.1006206
  72. Yang H, Zhang J, Zhang X, et al. CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus. Antiviral Res 2018;151:63-70. https://doi.org/10.1016/j.antiviral.2018.01.004
  73. Guo C, Wang M, Zhu Z, et al. Highly efficient generation of pigs harboring a partial deletion of the CD163 SRCR5 domain, which are fully resistant to porcine reproductive and respiratory syndrome virus 2 infection. Front Immunol 2019;10:1846. https://doi.org/10.3389/fimmu.2019.01846
  74. Wang H, Shen L, Chen J, et al. Deletion of CD163 exon 7 confers resistance to highly pathogenic porcine reproductive and respiratory viruses on pigs. Int J Biol Sci 2019;15:1993-2005. https://doi.org/10.7150/ijbs.34269
  75. Xu K, Zhou Y, Mu Y, et al. CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance. elife 2020;9:e57132. https://doi.org/10.7554/eLife.57132
  76. Tanihara F, Hirata M, Nguyen NT, et al. Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes. Anim Biotechnol 2021;32:147-54. https://doi.org/10.1080/10495398.2019.1668801
  77. Lillico SG, Proudfoot C, King TJ, et al. Mammalian interspecies substitution of immune modulatory alleles by genome editing. Sci Rep 2016;6:21645. https://doi.org/10.1038/srep21645
  78. Popescu L, Gaudreault NN, Whitworth KM, et al. Genetically edited pigs lacking CD163 show no resistance following infection with the ASFV isolate, Georgia 2007-1. Virology 2017;501:102-6. https://doi.org/10.1016/j.virol.2016.11.012
  79. Whitworth KM, Rowland RRR, Petrovan V, et al. Resistance to coronavirus infection in amino peptidase N-deficient pigs. Transgenic Res 2019;28:21-32. https://doi.org/10.1007/s11248-018-0100-3
  80. Xie Z, Pang D, Yuan H, et al. Genetically modified pigs are protected from classical swine fever virus. Plos Pathog 2018;14:e1007193. https://doi.org/10.1371/journal.ppat.1007193
  81. Van Breedam W, Delputte PL, Van Gorp H, et al. Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage. J Gen Virol 2010;91(Pt 7):1659-67. https://doi.org/10.1099/vir.0.020503-0
  82. Prather RS, Rowland RRR, Ewen C, et al. An intact sialoadhesin (Sn-SIGLEC1-CD169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus. J Virol 2013;87:9538-46. https://doi.org/10.1128/JVI.00177-13
  83. Zhang Q, Yoo D. PRRS virus receptors and their role for pathogenesis. Vet Microbiol 2015;177:229-41. https://doi.org/10.1016/j.vetmic.2015.04.002
  84. Whitworth KM, Rowland RRR, Ewen CL, et al. Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nat Biotechnol 2016;34:20-2. https://doi.org/10.1038/nbt.3434
  85. Burkard C, Opriessnig T, Mileham AJ, et al. Pigs lacking the scavenger receptor cysteine-rich domain 5 of CD163 are resistant to porcine reproductive and respiratory syndrome virus 1 infection. J Virol 2018;92:e00415-18. https://doi.org/10.1128/JVI.00415-18
  86. Whitworth KM, Prather RS. Gene editing as applied to prevention of reproductive porcine reproductive and respiratory syndrome. Mol Reprod Dev 2017;84:926-3. https://doi.org/10.1002/mrd.22811
  87. Alcami A, Carrascosa AL, Vinuela E. Interaction of African swine fever virus with macrophages. Virus Res 1990;17:93-104. https://doi.org/10.1016/0168-1702(90)90071-i
  88. Sanchez-Torres C, Gomez-Puertas P, Gomez-del-Moral M, et al. Expression of porcine CD163 on monocytes/macrophages correlates with permissiveness to African swine fever infection. Arch Virol 2003;148:2307-23. https://doi.org/10.1007/s00705-003-0188-4
  89. Palgrave CJ, Gilmour L, Lowden CS, Lillico SG, Mellencamp MA, Whitelaw CB. Species-specific variation in RELA underlies differences in NF-kappaB activity: a potential role in African swine fever pathogenesis. J Virol 2011;85:6008-14. https://doi.org/10.1128/jvi.00331-11
  90. Lillico SG, Proudfoot C, Carlson DF, et al. Live pigs produced from genome edited zygotes. Sci Rep 2013;3:2847. https://doi.org/10.1038/srep02847
  91. McCleary S, Strong R, McCarthy RR, et al. Substitution of warthog NF-κB motifs into RELA of domestic pigs is not sufficient to confer resilience to African swine fever virus. Sci Rep 2020;10:8951. https://doi.org/10.1038/s41598-020-65808-1
  92. Hubner A, Petersen B, Keil GM, Niemann H, Mettenleiter TC, Fuchs W. Efficient inhibition of African swine fever virus replication by CRISPR/Cas9 targeting of the viral p30 gene (CP204L). Sci Rep 2018;8:1449. https://doi.org/10.1038/s41598-018-19626-1
  93. Wang Q, Vlasova AN, Kenney SP, Saif LJ. Emerging and reemerging coronaviruses in pigs. Curr Opin Virol 2019;34:39-49. https://doi.org/10.1016/j.coviro.2018.12.001
  94. Song D, Moon H, Kang B. Porcine epidemic diarrhea: a review of current epidemiology and available vaccines. Clin Exp Vaccine Res 2015;4:166-76. https://doi.org/10.7774/cevr.2015.4.2.166
  95. Sun R, Leng Z, Zhai SL, Chen D, Song C. Genetic variability and phylogeny of current Chinese Porcine epidemic diarrhea virus strains based on spike, ORF3, and membranegenes. Sci World J 2014;2014:208439. https://doi.org/10.1155/2014/208439
  96. Hu S, Qiao J, Fu Q, et al. Transgenic shRNA pigs reduce susceptibility to foot and mouth disease virus infection. eLife 2015;4:e06951. https://doi.org/10.7554/eLife.06951
  97. Schmidt SM, Belisle M, Frommer WF. The evolving landscape around genome editing in agriculture: Many countries have exempted or move to exempt forms of genome editing from GMO regulation of crop plants. EMBO Rep 2020;21:e50680. https://doi.org/10.15252/embr.202050680
  98. Entine J, Felipe MSS, Groenewald JH, et al. Regulatory approaches for genome edited agricultural plants in select countries and jurisdictions around the world. Transgenic Res 2021;30:551-84. https://doi.org/10.1007/s11248-021-00257-8
  99. Van Enennaam AL, De Figueiredo Silva F, Trott JF, Zilberman D. Genetic engineering of livestock: The opportunity cost of regulatory delay. Annu Rev Anim Biosci 2021;9:453-78. https://doi.org/10.1146/annurev-animal-061220-023052
  100. Byrne J. New plant breeding techniques: EU Commission finds GMO legislation not fit for purpose [internet]; c2021 [2021 Apr 29]. Available from: https://www.feednavigator.com/Article/2021/04/29/EU-consultation-process-on-legalframework-for-NGTs-to-begin